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Pre-use integrity testing of sterilizing-grade filters eliminates the potential adverse effects of filter loading on the integrity-test results, allowing unambiguous correlation with the integrity-test specification established during filter-validation studies.
A septically produced liquid pharmaceutical and biopharmaceutical products are usually sterilized by filtration. The filtration process must be validated to ensure that it is capable of removing all microorganisms from the product. Validation consists of challenging the filter with a suspension of Brevundimonas diminuta and analyzing the filtrate for microorganisms. The filtrate must be sterile.
Filters used in production should be equivalent to the filters used in the bacterial-challenge validation studies. Because actual production filters cannot undergo bacterial-challenge testing, integrity testing is performed to demonstrate bacterial-retention equivalence. If the integrity-test values obtained for the production filters are equivalent to those obtained for the filters successfully passing bacterial-challenge testing, then it is assumed that the filters have the same bacterial-retention properties and that the filtered pharmaceutical product is, therefore, sterile.
Integrity testing for the hydrophilic filters used in pharmaceutical production relies on the measurement of gas flow through wetted membranes. This flow can be classified as diffusive or bulk and is sometimes a combination of both. Fick's Law of Diffusion shows that diffusion of the test gas through the liquid-filled pores in the membrane is a function of the diffusion constant and the solubility of the test gas in the liquid at the test temperature, the pressure differential of the test gas across the membrane, the thickness of the liquid layer, and the area and porosity of the membrane (1). Diffusion is not directly related to pore size although, as will be shown later, there is an indirect correlation. Bulk flow occurs when the test gas flows through the nonwetted or empty pores of the membrane. Open pores occur because the filter membrane has been incompletely wetted or because the bubble point of the membrane has been exceeded. Bulk flow primarily is a function of the size and number of the open pores, the thickness of the membrane, and the pressure differential of the test gas across the membrane at the test temperature.
Gas flow through wetted membranes
As indicated previously, gas flow through wetted membranes is diffusive, bulk, or a combination of both. Figure 1 shows the relationship between gas flow and differential pressure for a typical membrane filter. Similarly, because it is an arithmetic function, pure bulk flow is a straight line whose slope is determined by the size and number of open pores, assuming all other variables are held constant.
The knee area of the curve is where the influence of the bubble point is manifested. Here, the largest pores of the filter become unblocked as the applied pressure overcomes the capillary forces within those pores, and bulk flow begins to increase the slope of the curve. It is also in this region that diffusive flow begins to increase because the thickness of the liquid trapped in the largest pores begins to decrease as a result of the increasing pressure differential. Therefore, within the knee area of the curve a complex relationship exists between diffusive and bulk flow, both influenced by the pore structure and pore-size distribution.
Validating the bacterial-retention capabilities of sterilizing-grade filters involves challenging the filters with a suspension of B. diminuta (ATCC 19146) at a level of at least 1 × 107 CFU/cm2 of filter area, resulting in a sterile filtrate. Specific details and options may be found in "PDA Technical Report No. 26" (2). The integrity-test values of the filters used in the bacterial challenge studies must be known to form the basis for ensuring the filters used in production have equivalent bacterial-retention capabilities. In addition to the integrity-test values, the manufacturing processes and quality-assurance systems of the filter manufacturer must be adequate to ensure consistency of the filters within and between each manufactured lot with respect to nominal pore size, pore-size distribution, and membrane thickness and area.
Bacterial retention is assessed by challenging filters of the intended type having a range of integrity-test values and analyzing the bacterial-passage results. Ideally, the retention pattern observed will allow identification of physical integrity-test values above or below which, depending on the type of integrity test, there is virtually no probability of bacterial passage. For example, with a series of filters having a range of bubble points and equivalently challenged, there would be a value below which bacterial passage always occurred, a range where passage sometimes occurred, and a value above which passage never occurred.
It is important to identify the integrity values above and below which bacterial retention and bacterial passage can be expected. This ensures that the correlation between the integrity-test value and bacterial retention is valid and there are not other factors such as pore-size distribution and anisotropy that are potentially influencing the perceived correlation.
During development of what he referred to as the "forward flow" test in 1973, Dr. Pall performed bacterial-challenge testing on a series of 43 cartridges having water-wet forward air flow values ranging from 16 to 900 cm3/h at a pressure differential of 10 in of mercury (4.9 psi) (3). The results of this testing showed that no filter with a flow of less than 199 cm3/h allowed bacterial passage. On the basis of those results, he established a maximum flow limit of 100 cm3/h for an acceptable production filter element, building in a safety factor. However, the testing also showed that there were filters with forward-flow values of 210, 258, 450, and 645 cm3/h that retained the challenge organism and filters with forward-flow values of 199, 238, 471, and 600 cm3/h that allowed bacterial passage. The study did not establish a point above which bacterial passage always occurred; therefore the correlation between the integrity-test value and bacterial retention was not fully elucidated.
Because the forward-flow test as described relies on diffusive flow, which in terms of the filter membrane is a function of porosity, area, and thickness and is not directly related to pore size, the bacterial-retention results obtained for the higher forward-flow values could be indicative of pore-size distribution differences (or possibly thickness variations) among the tested membranes. Had these tests been carried out at pressures closer to the bubble point as is done today, differences in pore-size distribution might have been revealed as a result of the pressure-induced thinning of the liquid layer in the larger pores and better correlation obtained with bacterial retention at the higher diffusive-flow values. Nonetheless, Pall's efforts served as the basis for developing the automated filter-integrity tests in use today. These tests operate at considerably higher pressures, capturing the influence of the knee area and providing effective correlation between bacterial retention and the integrity-test value.
It is important to note that the integrity-test values were obtained on clean filters, before commencing the bacterial-retention tests, eliminating the influences of filter loading on filter porosity and diffusive-flow values.
Another validation consideration is to test the filters after sterilization and bacterial-challenge testing to evaluate the influences of filter loading and the sterilization process on the integrity-test values.
The sterilizing filtration process is designed to remove viable and nonviable particles from the fluid passing through the filter. The particles removed from the fluid either remain on the surface of the filter membrane or are trapped within the membrane matrix.
It has been shown that hydrodynamic flow is preferentially directed to the larger pores in a filter membrane (4). This means that hydrodynamic flow directs the suspended particles to the largest pores. However, because the largest pores are far less numerous than the average-sized pores, particles initially tend to be captured by the many smaller pores. As the smaller pores become blocked, the hydrodynamic flow directs more of the particles to the larger pores. The larger pores now may become blocked or partially blocked because of bridging, which is promoted by high levels of particulates competing for the pore openings. This phenomenon is corroborated by Grant and Zahka (5). They showed that during the filtration of a dilute suspension of silica particles there was complete retention at the beginning of the filtration, followed by particle passage. The retention then increased as the filtration continued. Similar results were obtained by Roberts and Velazquez while filtering latex particles (6). Thomas et al. found that the successive changes affecting a filter's pore-size distribution as a filtration of Pseudomonas aeruginosa progressed showed the larger pores to be less immediately engaged in the retention of particles (7).
Effect of filter loading on integrity-test results
The effects of filter loading on diffusive flow and the bubble point have been evaluated; however, comprehensive studies remain to be performed, and understanding of these effects is currently incomplete. Trotter et al. showed that the diffusive-flow values at 45 psi began to change at a bacterial cell loading of 1 × 102, while the bubble point remained essentially unchanged until the bacterial cell loading approached 1 x 108 (8).
Particle capture inherently reduces the porosity of the filter, thus decreasing the diffusive flow, and it increases the bubble point as the largest pores become blocked. Also, the effective thickness of the membrane increases as material accretes on its surface. These effects have implications with respect to the integrity-test values and their correlation to bacterial retention. Certainly, porosity reduction and the increase in thickness depend on the feed stream and the properties of the particles (gel or solid) removed from it.
Filter loading influences diffusive-flow and bubble-point values, although not to the same degree and not in the same sequence according to published studies. Postfiltration integrity testing evaluates filters that have been subject to an unknown level of particulate loading; therefore the influence of the particulate loading on the integrity-test results is also unknown. It is reasonable to assume, however, that as particles are arrested by the membrane, the porosity of the membrane decreases and the bubble point increases. This assumption has been confirmed experimentally (8). Testing under different conditions or with different membrane types and configurations could produce different results, however.
Extensive validation testing using multiple lots of product representative of normal production is necessary to determine the potential effects of filter loading on the postfiltration integrity-test values. In any case, the precise effect of the filter loading on the postfiltration integrity test cannot be known with certainty; therefore, it is not possible to ensure that filter loading has not masked a defect that initially may have been present in the filter.
Recommendations and conclusion
Regulatory agencies differ in their positions regarding the need for prefiltration integrity testing of membrane filters used to sterilize liquid pharmaceutical and biopharmaceutical products. Annex 1 of the EC Guide to Good Manufacturing Practice specifies integrity testing of the sterilized filter before and after use. FDA's 2004 Guidance for Industry, Sterile Drug Products Produced by Aseptic Processing suggests that integrity testing can be performed before processing and that integrity testing should be routinely performed post-use to detect leaks or defects that may have occurred during the filtration process. The FDA guidance does not take into account potential changes in the validated prefiltration integrity-test values as a result of filter loading.
Because the validation studies correlating bacterial retention with integrity-test results use prefiltration integrity-test values, albeit with a safety factor, the only reliable way to ensure that the filters used in production are equivalent to those used to establish the integrity-test specification is to perform a prefiltration integrity test. The postfiltration integrity test then serves its intended function: to ensure the filter has not been damaged during the sterilization or filtration process.
Russell E. Madsen* is president of the Williamsburg Group, LLC, 18907 Lindenhouse Rd., Gaithersburg, MD 20879, tel. 301.938.4266, firstname.lastname@example.orgMaik W. Jornitz is group vice-president of global product management, bioprocess of Sartorius Biotech Inc. Theodore H. Meltzer is a consultant for Capitola Consulting Company. Madsen and Meltzer are also members of Pharmaceutical Technology's Editorial Advisory Board.
*To whom all correspondence should be addressed.
Keywords: filtration, integrity testing. sterilization
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2. J.H. Robertson et al., "PDA Technical Report No. 26: Sterilizing Filtration of Liquids," PDA J. Pharm. Sci. Technol. 52 (S1), (1998).
3. D.B. Pall, "Quality Control of Absolute Bacteria Removal Filters," Bull. Paren. Drug Assoc. 29 (4), 192–204 (1975).
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6. K.L. Roberts and D.J. Velazquez, "Characterizing the Rating and Performance of Membrane Filters for Liqid Applications Using Latex Spheres," Swiss Contamination Control Quarterly 3 (4a), 71–74 (1990).
7. A.J. Thomas et al., "Detection of L-forms of Pseudomonas aeruginosa during Microbiological Validation of Filters." Pharm. Technol. 14(3), 34–44 (1992).
8. A.M. Trotter et al., "Investigation of a Filter Structure by Microbial Retention Studies: A Synthesis and Elaboration of Prior Findings," PDA J. Pharm. Sci. Technol. 55 (2), 127–133 (2001).